The present invention relates to the manufacture of isoprenoids using molecular biology techniques. In particular, the present invention provides DNAs, vectors and host cells for the efficient production of various enzymes in the mevalonate pathway or for converting isopentyl pyrophosphate to farnesyl pyrophosphate synthase.
Astaxanthin is reportedly distributed in a wide variety of organisms such as animals (e.g., birds, such as flamingo and scarlet ibis; fish, such as rainbow trout and salmon), algae and microorganisms. It is also reported that astaxanthin has a strong antioxidation property against oxygen radicals, which is believed to be pharmaceutically useful for protecting living cells against some diseases such as a cancer. Moreover, from a commercial prospective, there is an increasing demand for astaxanthin as a coloring reagent especially in the fish farming industry, such as salmon farming, because astaxanthin imparts a distinctive orange-red coloration to the fish and contributes to consumer appeal.
Phaffia rhodozyma is known as a carotenogenic yeast strain which produces astaxanthin specifically. Different from the other carotenogenic yeast, Rhodotorula species, such as Phaffia rhodozyma (P. rhodozyma) can ferment some sugars such as D-glucose. This is a commercially important feature. In a recent taxonomic study, the sexual cycle of P. rhodozyma was revealed and its telemorphic state was designated as Xanthophyllomyces dendrorhous (W. I. Golubev; Yeast 11, 101-110, 1995). Some strain improvement studies to obtain hyper-producers of astaxanthin from P. rhodozyma have been conducted, but such efforts have been restricted to conventional methods including mutagenesis and protoplast fusion in this decade.
Recently, Wery et al. reportedly developed a host vector system using P. rhodozyma in which a non-replicable plasmid was integrated into the genome of P. rhodozyma at the locus of a ribosomal DNA in multiple copies (Wery et al., Gene, 184, 89-97, 1997). Verdoes et al. reported vectors for obtaining a transformant of P. rhodozyma, as well as its three carotenogenic genes which code for the enzymes that catalyze the reactions from geranylgeranyl pyrophosphate to xcex2-carotene (International patent WO97/23633).
It has been reported that the carotenogenic pathway from a general metabolite, acetyl-CoA consists of multiple enzymatic steps in carotenogenic eukaryotes as shown in FIG. 1. In this pathway, two molecules of acetyl-CoA are condensed to yield acetoacetyl-CoA which is converted to 3-hydroxy-3-methyglutaryl-CoA (HMG-CoA) by the action of 3-hydroxymethyl-3-glutaryl-CoA synthas. Next, 3-hydroxy-3-methylglutaryl-CoA reductase converts HMG-CoA to mevalonate, to which two molecules of phosphate residues are then added by the action of two kinases (mevalonate kinase and phosphomevalonate kinase). Mevalonate pyrophosphate is then decarboxylated by the action of mevalonate pyrophosphate decarboxylase to yield isopentenyl pyrophosphate (IPP) which becomes a building unit for a wide variety of isoprene molecules which are necessary in living organisms. This pathway is designated the xe2x80x9cmevalonate pathwayxe2x80x9d taken from its important intermediate, mevalonate.
In this pathway, IPP is isomerized to dimethylaryl pyrophosphate (DMAPP) by the action of IPP isomerase. Then, IPP and DMAPP are converted to a C10 unit, geranyl pyrophosphate (GPP) by a head to tail condensation. In a similar condensation reaction between GPP and IPP, GPP is converted to a C15 unit, farnesyl pyrophosphate (FPP) which is an important substrate of cholesterol in animals, of ergosterol in yeast, and of the farnesylation of regulation proteins, such as the RAS protein. In general, the biosynthesis of GPP and FPP from IPP and DMAPP are catalyzed by one enzyme called FPP synthase (Laskovics et al., Biochemistry, 20, 1893-1901, 1981).
On the other hand, in prokaryotes such as eubacteria, isopentenyl pyrophosphate is reportedly synthesized in a different pathway via 1-deoxyxylulose-5-phosphate from pyruvate which is absent in yeast and animals (Rohmer et al., Biochem. J., 295, 517-524, 1993).
In studies of cholesterol biosynthesis, it was shown that the rate-limiting steps of cholesterol metabolism were in the steps of this mevalonate pathway, especially in its early steps catalyzed by HMG-CoA synthase and HMG-CoA reductase. It was recognized in accordance with the present invention that the biosynthetic pathways of cholesterol and carotenoid which share their intermediate pathway from acetyl-CoA to FPP can be used to improve the rate-limiting steps in the carotenogenic pathway. These steps may exist in the steps of mevalonate pathway, especially in the early mevalonate pathway such as the steps catalyzed by HMG-CoA synthase and HMG-CoA reductase. Improved yields of carotenoids, especially astaxanthin, are achievable using the process of the present invention.
In accordance with this invention, the genes and the enzymes involved in the mevalonate pathway from acetyl-CoA to FPP which are biological materials useful in improving the astaxanthin production process are provided. In the present invention, cloning and determination of the genes which code for HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, mevalonate pyrophosphate decarboxylase and FPP synthase is provided.
This invention also relates to the characterization of such enzymes as a result of the expression of such genes in suitable host organisms such as E. coli. These genes may be amplified in a suitable host, such as P. rhodozyma. The effects on carotenogenesis by these enzymes can be confirmed by cultivation of such a transformant in an appropriate medium under appropriate cultivation conditions.
In one embodiment, there is provided an isolated DNA sequence coding for at least one enzyme involved in the mevalonate pathway or the reaction pathway from isopentenyl pyrophosphate to farnesyl pyrophosphate. More specifically, such enzymes in accordance with the present invention are those having, for example, the following activities: 3-hydroxy-3-methylglutaryl-CoA synthase activity, 3-hydroxy-3-methylglutaryl-CoA reductase activity, mevalonate kinase activity, mevalonate pyrophosphate decarboxylase activity and farnesyl pyrophosphate synthase.
The isolated DNA sequences according to the present invention are more specifically characterized in that (a) they code for enzymes having amino acid sequences as set forth in SEQ ID NOs: 6, 7, 8, 9 and 10. The DNA sequences may alternatively (b) code for variants of such enzymes selected from (i) allelic variants and (ii) enzymes having one or more amino acid addition, insertion, deletion and/or substitution and having the stated enzyme activity.
Preferably, the isolated DNA sequence defined above is derived from a gene of Phaffia rhodozyma. Such a DNA sequence is represented in SEQ ID NOs: 1, 2, 4 and 5. This DNA sequence may also be an isocoding or an allelic variant for the DNA sequence represented in SEQ ID NOs: 1, 2, 4 and 5. In addition, this DNA sequence can be a derivative of a DNA sequence represented in SEQ ID NOs: 1, 2, 4 and 5 with addition, insertion, deletion and/or substitution of one or more nucleotide(s), and coding for a polypeptide having the above-referenced enzyme activity.
In the present invention, such derivatives can be made by recombinant means using one of the DNA sequences as disclosed herein by methods known in the art and disclosed, e.g. by Sambrook et al. (Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, second edition 1989) which is hereby incorporated by reference. Amino acid exchanges in proteins and peptides which do not generally alter the activity of the protein or peptide are known in the art and are described, for example, by H. Neurath and R. L. Hill in The Proteins (Academic Press, New York, 1979, see especially FIG. 6, page 14). The most commonly occurring exchanges are: Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly, as well as these in reverse.
The present invention also provides an isolated DNA sequence coding for a polypeptide having mevalonate kinase activity, which DNA is selected from (i) a DNA sequence represented in SEQ ID NO: 3; (ii) an isocoding or an allelic variant for the DNA sequence represented in SEQ ID NO: 3; and (iii) a derivative of a DNA sequence represented in SEQ ID NO: 3 with addition, insertion, deletion and/or substitution of one or more nucleotide(s).
The present invention is intended to include those DNA sequences as specified above and as disclosed in the sequence listing, as well as their complementary strands, DNA sequences which include these sequences, DNA sequences which hybridize under standard conditions with such sequences or fragments thereof and DNA sequences, which because of the degeneracy of the genetic code, do not hybridize under standard conditions with such sequences, but which code for polypeptides having exactly the same amino acid sequence.
For purposes of the present invention, xe2x80x9cstandard conditionsxe2x80x9d for hybridization mean the conditions which are generally used by a one skilled in the art to detect specific hybridization signals and which are described, e.g. by Sambrook et al., supra Preferably, the standard conditions are so called xe2x80x9cstringent hybridizationxe2x80x9d and non-stringent washing conditions, more preferably so called stringent hybridization and stringent washing conditions. The stringency (high vs. low) of a particular hybridization will of course vary depending upon, for example, the salt concentration and temperature of the hybridization and washes, as well as the lengths of the probe and target DNAs. The examples provided herein set forth representative hybridization conditions but are not to be construed as limiting the scope of the invention.
Furthermore, DNA sequences which can be made by the polymerase chain reaction using primers designed on the basis of the DNA sequences disclosed herein by methods known in the art are also included in the present invention. It is understood that the DNA sequences of the present invention may also be made synthetically as described, e.g. in EP 747 483 which is hereby incorporated by reference.
Another embodiment of the present invention is a recombinant DNA, preferably a vector and/or a plasmid including a sequence coding for an enzyme functional in the mevalonate pathway or the reaction pathway from isopentenyl pyrophosphate to farnesyl pyrophosphate. The recombinant DNA vector and/or plasmid of the present invention includes regulatory regions, such as for example, promoters and terminators, as well as open reading frames of above named DNAs.
Another embodiment of the present invention is a process for transforming a host organism with a recombinant DNA, vector or plasmid. The recombinant organism of the present invention overexpresses a DNA sequence encoding an enzyme involved in the mevalonate pathway or the reaction pathway from isopentenyl pyrophosphate to farnesyl pyrophosphate. The host organism transformed with the recombinant DNA is intended to be used for, e.g., producing isoprenoids and carotenoids, in particular astaxanthin. Thus the present invention also includes such recombinant organisms/transformed hosts.
Another embodiment of the present invention is a method for the production of isoprenoids or carotenoids, preferably carotenoids, which includes cultivating recombinant organisms containing a DNA construct coding for such isoprenoids or carotenoids.
Another embodiment of the present invention is a method for producing an enzyme involved in the mevalonate pathway or the reaction pathway from isopentenyl pyrophosphate to farnesyl pyrophosphate. This method includes culturing a recombinant organism as mentioned above, under conditions conducive to the production of the enzyme. The method may also relate to obtaining the purified enzyme itself.
Another embodiment is a process for overexpressing an enzyme in the mevalonate pathway or an enzyme in the pathway for converting isopentenyl pyrophosphate to farnesyl pyrophosphate. This process includes selecting at least one DNA sequence from the group consisting of SEQ ID NOs: 1-5; transforming a host cell culture with at least one of the DNA sequences selected; expressing in the host cell at least one enzyme in the mevalonate pathway or an enzyme in the pathway for converting isopentenyl pyrophosphate to farnesyl pyrophosphate; and recovering the enzyme(s) from the culture.
The present invention will be understood more easily on the basis of the enclosed figures and the more detailed explanations given below.